Pseudomonas aeruginosa Increases the Sensitivity of Biofilm-Grown Staphylococcus aureus to Membrane-Targeting Antiseptics and Antibiotics

Abstract

Pseudomonas aeruginosa and Staphylococcus aureus often cause chronic, recalcitrant infections in large part due to their ability to form biofilms. The biofilm mode of growth enables these organisms to withstand antibacterial insults that would effectively eliminate their planktonic counterparts. We found that P. aeruginosa supernatant increased the sensitivity of S. aureus biofilms to multiple antimicrobial compounds, including fluoroquinolones and membrane-targeting antibacterial agents, including the antiseptic chloroxylenol. Treatment of S. aureus with the antiseptic chloroxylenol alone did not decrease biofilm cell viability; however, the combination of chloroxylenol and P. aeruginosa supernatant led to a 4-log reduction in S. aureus biofilm viability compared to exposure to chloroxylenol alone. We found that the P. aeruginosa-produced small molecule 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) is responsible for the observed heightened sensitivity of S. aureus to chloroxylenol. Similarly, HQNO increased the susceptibility of S. aureus biofilms to other compounds, including both traditional and nontraditional antibiotics, which permeabilize bacterial membranes. Genetic and phenotypic studies support a model whereby HQNO causes an increase in S. aureus membrane fluidity, thereby improving the efficacy of membrane-targeting antiseptics and antibiotics. Importantly, our data show that P. aeruginosa exoproducts can enhance the ability of various antimicrobial agents to kill biofilm populations of S. aureus that are typically difficult to eradicate. Finally, our discovery that altering membrane fluidity shifts antimicrobial sensitivity profiles of bacterial biofilms may guide new approaches to target persistent infections, such as those commonly found in respiratory tract infections and in chronic wounds.IMPORTANCE The thick mucus in the airways of cystic fibrosis (CF) patients predisposes them to frequent, polymicrobial respiratory infections. Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from the airways of individuals with CF, as well as from diabetic foot ulcers and other wounds. Both organisms form biofilms, which are notoriously difficult to eradicate and promote chronic infection. In this study, we have shown that P. aeruginosa-secreted factors can increase the efficacy of compounds that alone have little or no bactericidal activity against S. aureus biofilms. In particular, we discovered that P. aeruginosa exoproducts can potentiate the antistaphylococcal activity of phenol-based antiseptics and other membrane-active drugs. Our findings illustrate that polymicrobial interactions can dramatically increase antibacterial efficacy in vitro and suggest that altering membrane physiology promotes the ability of certain drugs to kill bacterial biofilms-knowledge that may provide a path for the discovery of new biofilm-targeting antimicrobial strategies.

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; antibiotics; biofilm; membrane.

FIG 1

P. aeruginosa supernatant increases S. aureus biofilm sensitivity to chloroxylenol. Biofilm disruption assays on plastic were performed with the specified S. aureus clinical isolate, P. aeruginosa PA14 supernatant (Pa sup), and chloroxylenol at 100 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. Each column displays the average from two biological replicates, each with three technical replicates. Error bars indicate standard deviation (SD). Sa, S. aureus; bd, below detection; ns, not significant; **, P < 0.01; ***, P < 0.001, by ordinary one-way ANOVA and Bonferroni’s multiple-comparison posttest.

FIG 2

P. aeruginosa supernatant enhances the ability of chloroxylenol to kill difficult-to-treat S. aureus biofilms. (A) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, P. aeruginosa PA14 supernatant (Pa sup), and chloroxylenol (Chlor) at 100 μg/ml under normoxic or anoxic conditions. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. (B) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Col parental strain or hemB mutant, supernatants from wild-type P. aeruginosa PA14 and the ΔpqsL ΔpvdA ΔpchE mutant (Pa ΔΔΔ sup), and chloroxylenol (Chlor) at 100 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. (C) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, supernatants from wild-type P. aeruginosa PA14 and the ΔpqsL ΔpvdA ΔpchE mutant (Pa ΔΔΔ sup), and chloroxylenol (Chlor) at 100 μg/ml. Biofilms were grown for 24 h and exposed to the above treatments for 24 additional hours, and S. aureus biofilm CFU were determined. Each column displays the average from three biological replicates, each with three technical replicates. Error bars indicate standard deviations. bd, below detection; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.

FIG 3

The P. aeruginosa exoproducts HQNO and siderophores increase S. aureus biofilm and planktonic sensitivity to chloroxylenol. (A and B) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, supernatants from wild-type P. aeruginosa PA14 and the ΔpqsL ΔpvdA ΔpchE deletion mutant (Pa ΔΔΔ sup), and chloroxylenol (Chlor) at 100 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm (A) and planktonic (B) CFU were determined. Data in panels A and B were from the same experiments. (C) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, chloroxylenol (Chlor) at 100 μg/ml, and the specified concentrations of HQNO (dissolved in DMSO). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.

FIG 4

Exogenous HQNO increases S. aureus membrane fluidity. (A to C) Laurdan generalized polarization (GP) was performed with S. aureus (Sa) Newman, benzyl alcohol (BnOH) (A and B), HQNO (B), and the DMSO control (solvent for HQNO) (B) at the indicated concentrations and antimycin A at 100 μg/ml along with the ethanol (EtOH) control (solvent for antimycin A) (C). S. aureus was exposed to the above treatments for 1 h, and GP values were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.

FIG 5

Shifting membrane fluidity alters S. aureus biofilm sensitivity to chloroxylenol. (A to C) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, chloroxylenol (Chlor) at 100 μg/ml, benzyl alcohol (BnOH) at 50 mM (A), 1-heptanol at 50 mM (B), and dimethyl sulfoxide (DMSO) at 1% and 6% (C). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; **, P < 0.01; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.

FIG 6

P. aeruginosa supernatant increases S. aureus biofilm sensitivity to other membrane-targeting compounds. (A to E) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman; supernatants from wild-type P. aeruginosa PA14 and the specified mutants (Pa sup); and either biphenyl at 200 μg/ml (A), gramicidin at 100 μg/ml (B), trifluoperazine at 100 μg/ml (C), amitriptyline at 100 μg/ml (D), or octenidine dihydrochloride (Oct) at 5 μg/ml (E). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. Error bars indicate standard deviation (SD). ns, not significant; *, P < 0.05; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.